published_or_final_version / Anatomy / Master / Master of Medical Sciences

published_or_final_version / Community Medicine / Master / Master of Public Health

published_or_final_version / Community Medicine / Master / Master of Public Health

published_or_final_version / Community Medicine / Master / Master of Public Health

published_or_final_version / Dental Surgery / Master / Master of Dental Surgery

Section 1: Construction and screening of a genomic library for the mosquito Aedes aegypti (Diptera, culicidae). A genomic library has been constructed for this important vector of arboviral disease. Total genomic DNA and various classes of RNA from Ae. aegypti were used to screen this library. The results obtained indicate that this species has a short period interspersion pattern of repeated sequences. Transcription of these repeats could not be detected using total cytoplasmic RNA, hnRNA or mRNA as hybridisation probes. Section 2: Sequence organisation of ribosomal DNA in Aedes aegypti. The Aedes aegypti genomic library was used to isolate clones containing the intact ribosomal DNA (rDNA) repeat of this species. This has been restriction mapped and the transcribed regions have been identified. The rDNA repeat is 9.0 Kb in length and is present as approximately 500 head-to-tail tandemly repeated copies. A low level of intraspecies polymorphism of Ae. aegypti rDNA is evident. Two restriction polymorphisms have been identified within the rDNA repeat. Section 3: Analysis of ribosomal DNA variation within Ae. aegypti'and between closely related species. Four variant rDNA clones have been isolated. One of these' may contain the end of a tandem array of ribosomal genes. Another variant contains a duplication of rDNA within the internal transcribed spacer region of the ribosomal repeat. Sequence analysis of this clone has identified regions at the 3' end of the 18S rRNA gene of Ae. aegypti which show very strong homology with the corresponding regions in other species. Some repeated sequences have been identified downstream of the 18S rRNA gene in this clone. Preliminary analysis of the two other rDNA variants indicates that one contains a duplication or insertion of DNA in the 28S rRNA coding region and one contains non-transcribed spacer homologous sequences which are not associated with rRNA coding regions. Section 4: DNA probes for species identification of mosquitoes in the Anopheles gambiae complex. DNA sequences have been isolated which distinguish four of the morphologically identical members of the An. gambiae species complex. Two sequence classes were obtained. Class 1 homologues are highly reiterated in the genomes of An. arabiensis and An. merus, present in low copy number in An. melas and were not detected in An. gambiae s. s. Class 1 sequences are male specific in An. arabiensis. Class 2 homologues are highly reiterated in the genomes of An. merus and An. melas and present in low to middle copy number in An. gambiae s. s, and An. arabiensis. Sex specificity of Class 2 homologues does not occur in the species tested (An. gambiae s. s. and An. arabiensis). Hybridisation of these species specific DNA sequences to mosquitoes squashed directly onto nitrocellulose provides a simplified method of species identification.

Helicobacter pylori is regarded as the most important risk factor for peptic ulcer disease and gastric cancer. In Kurdistan region, northern Iraq, gastric cancer is rare (5/100,000). To investigate some possible reasons for this, the prevalence of H. pylori infection, gastric mucosal histopathological changes in H. pylori infected subjects, and virulence factor genotypes (especially dupA) of colonising strains were studied. The immune response to H. pylori infection, focusing on genes associated with T-helper (Th) and regulatory T-cell (Treg) cells, was also investigated. It was found that 79% of 163 adults and 37% of 120 children were seropositive for H. pylori (p<0.0001). For infected people, gastric lymphocyte infiltration was more prominent in the antrum (p=0.01). 71% of Iraqi H. pylori strains were positive for cagA and its presence was significantly associated with peptic ulcer disease (PUD) (p<0.01). cagA genes encoding four or more tyrosine phosphorylation motifs could not be found in any of the Iraqi strains. Isolates possessing the i1 form of vacA were significantly associated with GU (p<0.02). 32% of Iraqi H. pylori isolates were dupA-positive and presence of this gene was associated with PUD (p<0.01). The levels of IFNγ, IL-12 p35, IL-10, IL-4 and FOXP3 mRNA were found to be elevated in gastric mucosal samples from H. pylori-infected patients compared to those from H. pylori-negative patients (median increase 7-fold p=0.001; 17-fold p=0.002; 1320-fold p=0.001; 1184-fold p=0.001; and 3-fold p=0.01, respectively), indicating a predominant IL-4 and IL-10 (Th2) response. Interestingly, IFNγ mRNA levels were 16-fold higher in tissues taken from 17 infected smokers than found in tissues taken from 18 infected non-smokers (p=0.009). IL-4 mRNA levels in tissues from 20 infected females were 40-fold higher than in tissues from 15 males (p=0.005). Nucleotide sequencing of the dupA 3' region from 32 strains showed that dupA commonly had additional single base insertions or deletions that either truncated or extended the open reading frame (ORF). We have therefore classified dupA into two main groups: the common extended ORF within jhp0917-19 (dupA1), and dupA with an early stop codon to truncate the ORF (dupA2). ELISA performed on supernatants from H. pylori-infected gastric epithelial cell lines found no significant differences in IL-8 production between strains that possessed or lacked dupA. In comparison to wild-type H. pylori, disruption of dupA significantly reduced IL-12, IFNγ, TNFα and IL-8 production by peripheral blood mononuclear cells (PBMCs) in 2/4 strains. For the remaining 2 strains, where gene sequencing revealed a frame shift resulting in truncated dupA in the wild-type, the level of these cytokines was unchanged by dupA mutation. H. pylori infection is common in Kurdistan region and acquired at a young age. The low cancer rate may be partially explained by a predominant lymphocyte infiltration in the antrum rather than the corpus, which has been reported to be associated with reduced risk of gastric adenocarcinoma. An absence of the more toxic cagA genotype with four or more tyrosine phosphorylation motifs in the Iraqi strains, and the predominance of Th2 cytokine expression rather than a more pro-inflammatory Th1 response to H. pylori could also contribute to a reduced incidence of cancer. dupA1 appears to play an important role in promoting the inflammatory response of leukocytes to H. pylori.

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